A PPP-type pseudophosphatase is required for the maintenance of basal complex integrity in Plasmodium falciparum

During its asexual blood stage, P. falciparum replicates via schizogony, wherein dozens of daughter cells are formed within a single parent. The basal complex, a contractile ring that separates daughter cells, is critical for schizogony. In this study, we identify a Plasmodium basal complex protein essential for basal complex maintenance. Using multiple microscopy techniques, we demonstrate that PfPPP8 is required for uniform basal complex expansion and maintenance of its integrity. We characterize PfPPP8 as the founding member of a novel family of pseudophosphatases with homologs in other Apicomplexan parasites. By co-immunoprecipitation, we identify two additional new basal complex proteins. We characterize the unique temporal localizations of these new basal complex proteins (late-arriving) and of PfPPP8 (early-departing). In this work, we identify a novel basal complex protein, determine its specific role in segmentation, identify a new pseudophosphatase family, and establish that the P. falciparum basal complex is a dynamic structure.

Malaria is one of humanity's most ancient pathogens; so significant that hemoglobinopathies including thalassemias and sickle-cell trait persist because they provide protection from severe malaria [1][2][3] . In 2021, there were 240 million cases of malaria and about 627,000 deaths, mostly in children under five infected with Plasmodium falciparum 4 . Despite steady improvements, progress toward malaria eradication has slowed in recent years [4][5][6] . One significant hurdle toward malaria eradication is an incomplete understanding of fundamental parasite biology. P. falciparum is a member of the Apicomplexa, a divergent clade of obligate parasitic eukaryotes, and its genome bears little resemblance to model organisms with >50% of its genes lacking homology to known proteins 7,8 . Thus, highly conserved processes like cell division, are divergent in Plasmodium.
Asexual replication of P. falciparum in erythrocytes causes clinically relevant malaria symptoms. The parasite divides via schizogony, wherein the organellar contents for 20-36 daughter cells (merozoites) are generated within a common cytoplasm followed by segmentation, a partitioning of contents simultaneous with a synchronized cytokinesis [9][10][11][12] . Segmentation requires two multi-component protein structures known as the basal complex (BC) and the inner membrane complex (IMC) [13][14][15] . The IMC is a double membrane structure of flattened vesicles and proteins underlying the plasma membrane, providing structure to forming merozoites and anchoring the actinmyosin motor during invasion 14,[16][17][18] . In P. falciparum merozoites, the IMC is one continuous vesicle. A set of apical polar rings anchor the subpellicular microtubules [19][20][21] marking its apical end and the BC marks its basal end.
In the related Apicomplexan parasite, Toxoplasma gondii, the BC has been extensively studied and multiple proteins have been characterized [23][24][25][26]31,32 . In T. gondii, the class VI-like 33 myosin TgMyoJ and the centrin TgCen2 are hypothesized to mediate BC contraction 25,34 . Moreover, the T. gondii BC is dynamic, 25,35 with a well-constructed timeline of assembly and recruitment 31 .
Here, we identify and characterize PfPPP8-an essential Plasmodium falciparum BC protein and novel pseudophosphatase-a protein bioinformatically characterized as a phosphatase but possessing a mutation inhibiting enzymatic activity. Pseudophosphatases compose 14% of identified phosphatases and play varied roles, including regulating active phosphatases by temporarily sequestering phosphor-protein substrates 36,37 . Pseudophosphatases have been found in nearly every phosphatase family, but PfPPP8 is the first identified within the phosphoprotein phosphatase (PPP) serine-threonine phosphatase family 36 .
Using super-resolution, live cell, and expansion microscopy (Ultrastructure Expansion Microscopy (U-ExM)), we show that PfPPP8 is required to maintain BC integrity, but not for initial BC formation. We demonstrate the temporal localization of PfPPP8, which departs from the BC before contraction finishes, and identify and validate two additional BC proteins, PfMyoJ and PF3D7_0214700, recruited to the BC halfway through segmentation. This study establishes the Plasmodium BC as a dynamic structure and characterizes a novel, essential component.

Results
PfPPP8 is a novel, essential member of the Plasmodium falciparum basal complex PF3D7_1018200/PfPPP8 was identified via immunoprecipitation with the BC protein PfCINCH 15 . It has a transcriptional profile similar to PfCINCH's 38 , is predicted to be essential based on experiments in P. berghei and a transposon insertion screen in P. falciparum 39,40 , and has a predicted serine-threonine phosphatase domain 41 . To evaluate the localization of PfPPP8, we fused the spaghetti monster-V5 (smV5) 42 epitope tag to the C-terminus of PfPPP8 in the 3D7 parasite strain ( Supplementary Fig. 1a, b). By immunofluorescence, PfPPP8-smV5 colocalized with the BC proteins PfMORN1 and PfBCP1 (Fig. 1a), demonstrating PfPPP8 is a bona fide BC protein.
To determine if PfPPP8 was essential for asexual replication, we utilized the Tet repressor (TetR)-binding aptamer system 43 . In the PfPPP8-smV5 strain, we included ten copies of the TetR-binding aptamer in the 3' untranslated region and the TetR-DOZI fusion protein under the control of a constitutive Plasmodium promoter 43 (Supplementary Fig. 1a, b). In the presence of the TetR ligand, anhydrotetracycline (ATc), the TetR-DOZI fusion protein is released from the aptamer array and PfPPP8 is translated 43,44 . In the absence of ATc, TetR binds the Tet-aptamers and the fused PfDOZI sequesters target mRNA in stress granules preventing translation 43 (Supplementary Fig. 1c).
Because segmentation is paired with the final karyokinesis in Plasmodium 12 , we wanted to see whether PfPPP8-knockdown modified the localization or distribution of the parasite centrosomes. There was, however, no significant difference in the appearance of the centrosome between PfPPP8-sufficient and -deficient parasites in slide-based or batch-stained IFAs ( Supplementary Fig. 3a, b). Furthermore, the division of nuclei in PfPPP8-deficient parasites was comparable to PfPPP8-sufficient parasites ( Supplementary Fig. 3a, b).

PfPPP8 is essential for the stability of the basal complex
Immunofluorescence showed that in PfPPP8-deficient parasites, BC proteins PfMORN1 and PfBCP1 formed fragmented or nonuniform rings ( Supplementary Fig. 4a) 15,22 . We tagged PfPPP8-interacting BC protein PfCINCH with the spaghetti monster-Myc (smMyc) 42 epitope tag in the PfPPP8-smV5 Tet background, generating PfPPP8-smV5 Tet ; PfCINCH-smMyc parasites. Visualizing PfCINCH-smMyc in PfPPP8deficient parasites allowed us to clearly see BC rings with multiple breakage points, fragmented BC rings, and BC rings that varied significantly in size and shape within individual parasites, compared to the uniform BC rings of PfPPP8-sufficient parasites (Fig. 2a). When we tagged the BC protein PfBLEB in the PfPPP8-smV5 Tet background 29 with HaloTag 50 (generating the PfPPP8-smV5 Tet ; PfBLEB-HaloTag line) we also saw disorganized, fragmented PfBLEB-HaloTag rings in PfPPP8deficient parasites, confirming PfPPP8 is essential for BC stability ( Supplementary Fig. 4b).
To quantify differences in BC shape between PfPPP8-sufficent and -deficient parasites, and within individual PfPPP8-deficient parasites, we calculated the circularity of each visible BC ring in ten parasites per condition, using PfCINCH-smMyc as a BC marker. This measure (where proximity to 1 on a 0-1 scale indicates a more circular shape) allowed us to compare the jagged, irregular BCs of PfPPP8-deficient parasites to more uniform, rounder PfPPP8-sufficient BCs (Fig. 2b, c). The mean circularity of BC rings in PfPPP8-deficient parasites was significantly lower (0.71 ± 0.12) than that of PfPPP8-sufficient parasites (0.82 ± 0.07) (p < 0.0001), indicating defects in BC expansion or organization (Fig. 2b, c). Circularity measurements for individual parasites (Fig. 2b) also demonstrate that PfPPP8-deficient parasites have a greater range of circularity values (0.6954 vs 0.3815 for PfPPP8-sufficient), and an Ftest confirms their variance is significantly greater (p < 0.0001). Thus, synchronous, uniform BC expansion is compromised in PfPPP8deficient parasites.
Next, we utilized U-ExM 51 with a non-specific fluorescent protein stain (N-hydroxysuccinimide ester linked to AlexaFluor 405 [NHS-AF405]) 52 , recently used to visualize the P. falciparum conoid 51,53 . In PfPPP8-smV5 Tet ; PfCINCH-smMyc parasites, NHS-AF405 clearly delineates the BC, validated by anti-Myc and anti-V5 co-staining. In Fig. 1 | PfPPP8 is a basal complex protein required for the formation of uniform merozoites during segmentation. a Localization of PfPPP8 via immunofluorescence and colocalization with PfBCP1 (left images) and PfMORN1 (right images). Top row, individual slices; bottom row, maximum projections. b PfPPP8-smV5 Tet replication curve with (PfPPP8-sufficient) and without (PfPPP8-deficient) ATc. Data shown as mean with 95% CI (n = 3). c Quantification of PfPPP8 knockdown by immunoblot. Data shown as mean ± SD (n = 4). d Visualization of PfPPP8deficient phenotype with an inner membrane complex marker (PfGAP45) and apical organellar marker (PfAMA1) compared to PfPPP8-sufficient parasites mid-segmentation and stalled with E64 via immunofluorescence. e Visualization of PfPPP8-deficient phenotype with a parasite plasma membrane marker (PfMSP1) and a basal complex marker (PfMORN1) compared to PfPPP8-sufficient parasites mid-segmentation and stalled with E64 via immunofluorescence. f Visualization of PfPPP8-deficient phenotype via transmission electron microscopy of E64-stalled schizonts, demonstrating overall defective segmentation despite properly formed rhoptries (red arrow) and apical polar rings (blue arrow). White boxes on the image of the entire schizont (left image in each column) indicate the zoomed-in region depicted in the right image of each panel. Each violin represents a set of circularity values for a single parasite's basal complex rings (n = 12). c Quantification of basal complex circularity in grouped PfPPP8sufficient and -deficient parasites; two-tailed unpaired Welch's t-test (p < 0.0001, n = 230 BC rings). Note that measurements of circularity do not utilize units and are always between 0 (least circular) and 1 (a perfect circle). d PfPPP8-sufficient and -deficient basal complex phenotype visualized by U-ExM. Top two rows show two individual slices from mid-segmentation PfPPP8-sufficient and PfPPP8-deficient schizonts; each slice is shown with only the epitope-specific fluorescent channels on the left and the fluorescent channels plus NHS-Ester on the right. Below, maximum projections of both parasites. Adjacent to each are images of single rings, isolated for emphasis and corresponding to the boxed areas of individual slices (boxes 1-3, PfPPP8-sufficient, boxes 4-6, PfPPP8-deficient).
Via U-ExM, the BC of PfPPP8-deficient parasites is extensively fragmented. Breaks in the BC are visible both by PfCINCH-smMyc and NHS-AF405-thus, these breaks separate the entire complex, rather than individual proteins ( Fig. 2d and Supplementary Movie 2). Max projections show each BC "ring" has multiple breaks; unlike in regular IFA, no intact (lacking breaks) BC rings are visible by U-ExM. PfPPP8deficient BC rings showed variably sized and extensively fragmented BC rings in three-dimensional structured illumination microscopy as well ( Supplementary Fig. 5a). Immunoblotting of PfPPP8-smV5 Tet ; PfCINCH-smMyc parasites showed PfPPP8-deficient parasites had PfMORN1/ PfCINCH levels comparable to PfPPP8-sufficient parasites, suggesting that increased BC fragmentation is not due to lack of expression of other BC proteins ( Supplementary Fig. 5b, c).

PfPPP8 is not required for initial BC biogenesis
At the achievable level of knockdown, young, small BC rings of PfPPP8deficient early segmentation stage schizonts appeared uniform but never maximally expanded or contracting rings ( Fig. 3a-d). We hypothesized that BC fragmentation in PfPPP8-deficient parasites could be time-dependent, based on the stage of segmentation. To evaluate this, we synchronized PfPPP8-smV5 Tet ; PfCINCH-smMyc parasites within a 2-h window. The BC was visualized via immunofluorescence every 2 h during segmentation (40-48 hpi). For ≥6 parasites per time point and condition, the diameter of every ring (intact or with a ≤0.4 µm break) was measured, totaling >100 each (Representative parasites: Fig. 3a). During initial BC formation (40 hpi), there was no significant difference between PfPPP8-sufficient and -deficient parasites in mean BC diameter (BCD) or BCD variance.
We then optimized a protocol for long-term live cell fluorescent imaging in P. falciparum to visualize BC formation, expansion, and contraction 50,54 . Using PfPPP8-smV5 Tet ; PfBLEB-HaloTag, we took 3-dimensional images every 20 min and measured the diameter of each BC ring in pairs of parasites matched by initial mean BCD (Fig. 3c BCD variance in the PfPPP8-deficient parasite started to increase at TP 1:20, 1 h and 20 min after TP 0:00, when the PfPPP8-sufficient parasite's mean BCD was 0.79 ± 0.07 µm. From time point (TP) 2:00 to TP 2:20, the BCD standard deviation in the PfPPP8-deficient parasite increased from 0.086 to 0.18 µm; its BC rings expanded nonuniformly (Fig. 3c, d). In the PfPPP8-sufficient parasite, the BCD standard deviation increased from 0.055 to 0.073. At TP 2:20, the PfPPP8-sufficient parasite's BCD was greatest (1.07 ± 0.07 µm) and their difference in BCD variance became statistically significant (p = 0.0005). The PfPPP8deficient parasite had a 1.23 µm range in BCD at TP 2:20, almost 7-fold higher than the PfPPP8-sufficient parasite's (range: 0.177 µm). As the PfPPP8-sufficient parasite's BC contracted, the PfPPP8-deficient parasite's BCD variance increased (p < 0.0001 from TP 2:40 onward) (Fig. 3c, d). BCD variance in the PfPPP8-deficient parasite remained significantly larger than in the PfPPP8-sufficient parasite (p < 0.0001) (Fig. 3c, d). These data corroborate fixed immunofluorescence timeline data; PfPPP8 is required to maintain BC integrity and uniform expansion.

PfPPP8 is a founding pseudophosphatase within the PPP family
Automated bioinformatic analysis identified a PPP-type serine-threonine protein phosphatase domain in PfPPP8 7 , but manual investigation of its sequence revealed that catalytic residues of one core motif (GDXXDRG) are mutated (GNLINRG) ( Supplementary Fig. 6a, b) 41,55-57 . These aspartic acids coordinate metal ions [58][59][60] , and their alteration disrupts phosphatase activity 61,62 . PfPPP8 is thus a pseudophosphatase -a protein with a phosphatase domain but lacking enzymatic activityand the first pseudophosphatase identified in the PPP family 36 .
PfPPP8 homologs in Cryptosporidia parvum, Babesia bovis, and Theileria parva have been identified in silico 63,64 . We used these homologs as seeds for protein-BLAST searches of PiroPlasmDb, EuPathDb, PlasmoDB, and ToxoDB, and investigated extant literature 31,63,65,66 . Homologs were identified across and beyond Apicomplexa (Supplementary Table 1). All contained bioinformatically predicted serine-threonine protein phosphatase domains 56,57 (Supplementary Fig. 6b). Most homologs possessed mutations in critical catalytic motifs, but even those with seemingly intact motifs like TgBCC5 had different numbers of amino acids between them than PPP-type phosphatase S. cerevisiae PPT1 (Supplementary Fig. 6b and Supplementary Note 1) 7,31,65,67,68 . PfPPP8 orthologs were also found in freeliving protists Vitrella brassicaformis and Chromera velia which share a common ancestor with Apicomplexans-thus, despite significant differences in lifestyle and a dearth of knowledge about the cell biology of colpodellids, mechanisms of division are likely to be similar even if, as with T. gondii and BCC5, PfPPP8's homolog does not play an identical role (Supplementary Table 1) 65,69,70 .
Some pseudophosphatases can attain phosphatase activity when catalytic residues are restored 37,71 . We recombinantly expressed phosphatase domains of PfPPP8 WT, PfPPP8 N1311,1314D , CpPPP8, CpPPP8 G406D,445R and S. cerevisiae PPT1 with C-terminal 6xHis-tags (Supplementary Fig. 6c) and, separately, with N-terminal maltosebinding protein (MBP)-tags, which generated a larger amount of more pure recombinant protein ( Supplementary Fig. 6d). Both 6xHis-ScPPT1 and MBP-ScPPT1 dephosphorylated p-nitrophenyl phosphate but no recombinant Apicomplexan phosphatase domains could, regardless of catalytic residue identity ( Supplementary Fig. 6e, f) 72 . Considering the differences in the number of amino acids present between conserved motifs between PfPPP8 (and other Apicomplexan homologs) and ScPPT1, this family of pseudophosphatases could potentially lack activity because they differ structurally from active Ser/Thr phosphatases in ways that preclude activation by sequence changes alone (Supplementary Note 1).

PfPPP8 interacts with other members of the basal complex including PfMyoJ and PF3D7_0214700
Because few Plasmodium BC proteins have been validated, we wanted to identify more components of this multi-protein molecular machine 31,32 . Tightly synchronized PfPPP8-smV5 Tet ; PfCINCH-smMyc parasites (and the parental strains) were collected at midsegmentation and subjected to co-immunoprecipitation. Following two biological replicates, we identified and compared consensus proteins for PfPPP8-V5 and PfCINCH-Myc (Fig. 4a, b).
Almost 50 proteins were shared between the consensus lists (Supplementary Table 2). From the proteins enriched >50-fold compared to the control, we selected two for validation ( Fig. 4b and Supplementary Table 2): PF3D7_1229800/PfMyoJ, because its homolog, TgMyoJ, is in the T. gondii BC 31,34,73 and PF3D7_0214700 because it was uncharacterized and unique to Plasmodium (and Hepatocystis) 65 . While examining PfMyoJ could identify conserved segmentation mechanisms between Apicomplexans and was likely to be localized to the BC, we were interested in PF3D7_0214700 because of its limitation to Plasmodium/Hepatocystis spp. As a unique protein, characterization of PF3D7_0214700 could elucidate mechanisms of segmentation, aspects of BC composition, and BC functions specific to the sub-Apicomplexan Plasmodiidae family 10 . Furthermore, as one of the smallest yet-uncharacterized proteins we identified (35kD), PF3D7_0214700 lends itself readily to future in-vitro experimentation (Supplementary Table 2). We thus generated C-terminally tagged PfMyoJ-smV5 and PF3D7_0214700-smV5 strains; both colocalized with PfMORN1, validating their identification as BC proteins (Fig. 4c, d).
Since both new proteins are recruited to the maximally expanded BC, we wanted to determine if they could be recruited to the morphologically defective BC of PfPPP8-deficient schizonts and generated the lines: PfPPP8-smV5 Tet ; PfMyoJ-smMyc and PfPPP8-smV5 Tet ; PF3D7_0214700-smMyc. PfMyoJ-smMyc colocalized with PfPPP8-smV5 and PfMORN1 in both PfPPP8-sufficient and -deficient parasites, the latter demonstrating that PfMyoJ was still recruited to the BC (Supplementary Fig. 8a). PfMyoJ remained at the IMC's leading edge in late segmentation PfPPP8-deficient schizonts, localizing to the disorganized, fragmented BC ( Supplementary Fig. 8b). PF3D7_0214700 was also still recruited to the fragmented, nonuniform BC of PfPPP8-deficient parasites, colocalizing with remaining PfPPP8-smV5 and localizing to the edge of the IMC (Supplementary Fig. 8c).
To corroborate this, we generated PfCINCH-mNeon-Green; PfPPP8-mCherry parasites and imaged a mixed-schizont population over 5 h. PfPPP8 and PfCINCH's temporal expressions were compared by examining adjacent schizonts that began imaging at different stages of segmentation-the left early, the right later ( Fig. 6c and Supplementary Movie 7). We determined the mean fluorescence intensity (MFI) for each fluorophore at each time point and plotted the relative change in each protein's MFI compared to TP0. PfCINCH-mNeonGreen's MFI increased during contraction, inversely correlated with ring diameter, as did the MFI of late segmentation PfPPP8-smV5 Tet ; PfBLEB-HaloTag parasites ( Fig. 6c and Supplementary Fig. 9d, f). In contrast, PfPPP8-mCherry's MFI decreased significantly during contraction, consistently across replicates (Fig. 6c, d, Supplementary  Fig. 11c, d and Supplementary Movies 7 and 8).

Discussion
We determined that PfPPP8, a novel BC protein in P. falciparum, is essential for maintaining BC integrity and uniformity. With U-ExM we confirmed that in PfPPP8-deficient schizonts, BC fragmentation was extensive throughout the entire BC; U-ExM revealed breakages too small to distinguish via conventional immunofluorescence.
There is increased intra-parasite BCD variance in PfPPP8deficient parasites after 44 hpi. Their BCs do not uniformly expand; evident when the BCD reaches 0.7-0.85 microns. Long-term, live cell microscopy of individual parasites showed this significant increase in intra-parasite BCD variance by mid-segmentation, with breakages accumulating throughout segmentation. PfPPP8-sufficient parasites maintained low intra-parasite BCD variance. While a more complete PfPPP8 knockout could potentially abrogate initial complex formation, PfPPP8 is~85% depleted upon knockdown, and many attempts to generate an inducible knockout have been unsuccessful.
PfPPP8 was also identified as a pseudophosphatase-the first experimentally characterized PPP-type serine-threonine pseudophosphatase and one of few Plasmodium pseudophosphatases 77 . Neither PfPPP8 nor its Cryptosporidium homolog were, or became through restoration of catalytic residues, enzymatically active. However, even PfPPP8's homologs without obvious mutations in catalytic motifs (TgBCC5 and VbPPP8) had more amino acids between motifs than active PPP-type phosphatase ScPPT1. This Apicomplexan pseudophosphatase family could be significantly diverged from active phosphatases, incapable of being easily rendered active as a result of larger structural changes to the phosphatase domain(s) 36 .
Whether most of PfPPP8's homologs function similarly in asexual division remains unknown. However, TgBCC5 was proven nonessential to Toxoplasma endodyogeny -TgBCC5-KO parasites are only deficient in intravacuolar communication between tachyzoites, a significant divergence from PfPPP8's importance to schizogony 31,70 . Furthermore, TgBCC5 is present in mature tachyzoites' BC, whereas PfPPP8 is degraded before schizogony ends 70 . TgBCC4-deficient parasites do resemble PfPPP8-deficient schizonts, surprisingly, since TgBCC4 is structurally distinct from PfPPP8 and lacks Plasmodium homologs. TgBCC4 also leaves the T. gondii BC before TgCEN2/TgMyoJ recruitment, unlike PfPPP8 which departs before egress, but after PfMyoJ recruitment 31 . Likely, the coordinated construction of dozens of BCs in schizogony requires novel and/or specially adapted protein(s) for maintaining both stability and synchrony.
In T. gondii, recent studies have suggested a model where the BC expands and stretches like a rubber band until it reaches its maximal diameter at the midpoint of segmentation 26,31 . PfPPP8deficient schizonts' increased BCD mean and variance suggests they exceed this "maximal stretch", resulting in BC fragmentation-the rubber band "snaps". PfPPP8 could thus act to signal this "maximal stretch" point. Aside from limiting BC "stretch" to a consistent maximal diameter, PfPPP8 is essential for uniform BC expansion. In Plasmodium schizogony, BCs expand like multiple rubber bands simultaneously stretched to the same degree. This requires communication between developing merozoites, linking PfPPP8 functionally to TgBCC5 and intravacuolar communication between tachyzoites 70 . Since T. gondii cytokinesis happens in cycles rather than simultaneously, this communication is less necessary and TgBCC5 less essential than PfPPP8, although they may be acting in similar processes.
The presence of PfPPP8 homologs across Apicomplexa indicates an ancient origin and a broad importance to multiple division modalities. Endopolygeny in Cystoisospora, for example, requires the coordination of dozens of BCs, like schizogony 10 . PfPPP8 homologs in photosynthetic, free-living protists suggest that the BC as a cytokinetic structure evolutionarily predates parasitism, and a PfPPP8-like protein could be essential to the "original" BC's formation.
PfPPP8's structure allows us to speculate about potential mechanisms. Although two Asp to Asn mutations render PfPPP8 catalytically inactive, its PPP-type phosphatase domain is otherwise intact. The negatively charged asparagine residues mutated in PfPPP8 coordinate calcium ions during dephosphorylation in active PPP-type phosphatases 58 . Since the positively charged amino acids in other motifs have not been modified, PfPPP8 could theoretically bind phosphorylated protein(s) in early segmentation, releasing it upon depletion as part of the tightly regulated cascade of events leading to egress. Indeed, C. elegans pseudophosphatases EGG-3 and EGG-4/5 bind and sequester kinase MBK-2, controlling its activity during the oocyte to zygote transition 78,79 . MBK-2 is released only when EGG-3 is degraded 80 . PfPPP8 could similarly function to temporarily "trap" phosphorylated substrate(s) until its degradation during late segmentation.
Dual co-immunoprecipitation identified PfMyoJ and PF3D7_0214700 as BC proteins. PfMyoJ's localization to the BC and late recruitment mimics TgMyoJ. While PfMyoJ was predicted to be nonessential and demonstrated to be nonessential for asexual blood stages in P. berghei, TgMyoJ-KO tachyzoites have incomplete BC contraction. PfMyoJ may similarly contribute to, despite not driving, BC contraction 34,40,81 . PF3D7_0214700 has a similar temporal localization, though it lacks identifiable domains and Toxoplasma homologs 7,40,56 . It is predicted to be essential and could be a useful phenotypic comparand for PfPPP8 and PfCINCH. Interestingly, both proteins still localize to the BC in PfPPP8-deficient schizonts, indicating that synchronous expansion is not necessary for later recruitment-considering BC breakage points appear frequently in 46 hpi schizonts, not even an intact BC is necessary. This indicates also that PfPPP8 does not comprise an essential part of the signal for PfMyoJ/Pf3D7_0214700 recruitment. Though TgBCC4's absence also induces fragmentation of the Toxoplasma BC, whether this fragmentation hampers TgMyoJ and TgCen2 recruitment remains to be determined.
PfPPP8's temporal localization contrasts with PfMyoJ and PF3D7_0214700's. U-ExM and long-term live cell imaging determined that PfPPP8's departure from the BC begins during contraction. PfPPP8's decrease in MFI contrasts with the concurrent MFI increase of PfBLEB-HaloTag and PfCINCH-mNeonGreen. Without protein depletion, this inverse relationship between BCD and MFI is expected as the same amount of protein is packed into a now-smaller area. When relative MFIs of PfCINCH-mNeonGreen, PfBLEB-HaloTag, and PfPPP8-mCherry in contracting BCs are averaged, linear regression returns a positive slope for PfCINCH-mNeonGreen and PfBLEB-HaloTag, and a negative slope for PfPPP8-mCherry. The pattern of PfCINCH's MFI increasing and PfPPP8's decreasing during contraction holds true when the fluorophores are switched; PfPPP8-mNeonGreen's mean fluorescence decreases during contraction just as PfPPP8-mCherry's does, strongly corroborating previous evidence.
Identification of unique, disparate temporal localizations for all 3 proteins establishes the P. falciparum BC as a dynamic structure. Proteins including PfMORN1, PfBCP1, and PfCINCH are present in the BC for segmentation's duration. Proteins including PF3D7_0214700 and PfMyoJ are recruited in mid-segmentation, and proteins including PfPPP8 are present in the BC in early segmentation but depart before contraction is complete, a model depicted in Supplementary Fig. 13.
In conclusion, we identified PfPPP8 as an essential member of the P. falciparum BC, required for maintaining uniformity and integrity. We also identified PfPPP8 as the founding member of a PPP-type pseudophosphatase family with homologs throughout and beyond Apicomplexa. We identified PfMyoJ and PF3D7_0214700 as members of the BC and, along with PfPPP8, identified their unique temporal localizations. This establishes a strong foundation for further P. falciparum BC studies determining the order of assembly and disassembly of proteins within the BC.
PfCINCH-smMyc (pAM27). SmMyc was PCR amplified with oJDD4293/4925 and cloned as a NcoI/XmaI fragment into pRR206. The hDHFR cassette in this intermediate plasmid was removed by excision with AflII/AvrII and replaced with a blasticidin-S-deaminase expression cassette. The PfCINCH-targeting regions from pRR92 were excised with NotI/NcoI and cloned into the second intermediate plasmid using the same sites.
PfPPP8-mCherry (pAM110). The 5' half of mCherry was amplified with oJDD6503/6504. The 3' half of mCherry was amplified with oJDD6505/ 6506. The two fragments were spliced by PCR SOEing with oJDD6503/ 6506, removing an internal NcoI site, then cloned as a NcoI/KpnI fragment into pAM27. The PfPPP8-targeting regions from pRR206 were then excised with NotI/NcoI and cloned into the intermediate plasmid using the same sites.
PfMyoJ-smV5 Tet (pAM145). The 3' HR and 5'HR of PfMyoJ were PCR amplified from 3D7 gDNA with oJDD6358/6359 and oJDD6360/6361, respectively. A codon-altered region consisting of the last 70 amino acids was generated by IDT DNA technologies, amplified using oJDD6362/6363, and sewed onto the 5'HR by nested PCR with oJDD6360/6363. The joined fragments were spliced by PCR SOEing with oJDD4692/4690 and cloned as a NotI/NcoI fragment into pRR92 15 .
CpPPP8[AAs 370-683] G406D, G445R -MBP (pAM140). The 5' region of the phosphatase domain of CpPPP8 [cgd2_1640] was amplified from pAM139 with oJDD6849/6870 and the 3' region was amplified from pAM139 with oJDD6850/6871, containing modifications to change amino acids G406 and G445 to D and R respectively. Fragments were spliced together PCR-SOEing and cloned into pJDD283, digested with EcoRI and HindIII, using the NEBuilder HiFi DNA assembly cloning kit.

Reagents and antibodies
Primers were obtained from IDT (Integrated DNA Technologies) or Life Technologies. The sequence of each primer used is listed in Supplementary Table 3 Research, mouse anti-PfMSP1, clone 1E1, from Anthony Holder at MRC National Institute for Medical Research, mouse anti-PfLDH from Michael Makler at Flow Inc. Primary rabbit antisera against PfMORN1 and PfBCP1 were previously generated in the lab 15 . The 646 nm JaneliaFluor HaloTag ligand was obtained from Promega.

P. falciparum transfection
To construct the lines PfPPP8-smV5 Tet , PfMyoJ-smV5, and PF3D7_0214700-smV5, 100 μg of HDR plasmid was linearized by digestion, purified, and co-transfected with 75 μg Cas9 targeting plasmid into the P. falciparum 3D7 strain. Lines were maintained with 500 nM ATc from the onset of transfection; 1 day post transfection, drug pressure was applied with 2.5 nM WR99210 (Jacobus Pharmaceuticals).
To construct the line PfPPP8-smV5 Tet ; PfCINCH-smMyc, 100 μg of HDR plasmid was linearized by digestion, purified, and co-transfected with 75 μg Cas9 targeting plasmid into the PfPPP8-smV5 Tet strain. The line was maintained with 2.5 nM WR99210 and 500 nM ATc; 1 day post transfection, additional drug pressure was applied with 2.5 nM Blastocidin (Research Products International).
To construct the line PfPPP8-smV5 Tet ; PfBLEB-HaloTag, 100 μg of HDR plasmid was linearized by digestion, purified, and co-transfected with 75 μg Cas9 targeting plasmid into the PfPPP8-smV5 Tet strain. The line was maintained with 2.5 nM WR99210 and 500 nM ATc; 1 day post transfection, additional drug pressure was applied with 2.5 nM Blastocidin (Research Products International).
To construct the line PfPPP8-smV5 Tet ; PfMyoJ-smMyc, 100 μg of HDR plasmid was linearized by digestion, purified, and co-transfected with 75 μg Cas9 targeting plasmid into the PfPPP8-smV5 Tet strain. The line was maintained with 2.5 nM WR99210 and 500 nM ATc; 1 day post transfection, additional drug pressure was applied with 2.5 nM Blastocidin (Research Products International).
To construct the line PfPPP8-smV5 Tet ; PF3D7_0214700-smMyc, 100 μg of HDR plasmid was linearized by digestion, purified, and cotransfected with 75 μg Cas9 targeting plasmid into the PfPPP8-smV5 Tet strain. The line was maintained with 2.5 nM WR99210 and 500 nM ATc; 1 day post transfection, additional drug pressure was applied with 2.5 μg/ml blasticidin (Research Products International).
To construct the line PfCINCH-mNeonGreen; PfPPP8-mCherry, 100 μg of HDR plasmid containing the mNeonGreen fluorescent tag fused to the 3' end of the PfCINCH ORF (pRR208) was linearized by digestion, purified, and co-transfected with 75 μg Cas9 targeting plasmid into the P. falciparum 3D7 strain. One day post transfection, drug pressure was applied with 2.5 nM WR99210 (Jacobus Pharmaceuticals). Following verification of PfCINCH-mNeonGreen integration, PfCINCH-mNeonGreen parasites were transfected with 100 μg HDR plasmid containing the mCherry fluorescent tag fused to the 3' end of the PfPPP8 ORF (pAM110), linearized by digestion, and 75 μg Cas9 targeting plasmid. One day post transfection, additional drug pressure was applied with 2.5 nM Blastocidin (Research Products International).
To construct the line PfCINCH-mScarlet; PfPPP8-mNeonGreen, 100 μg of HDR plasmid containing the mScarlet fluorescent tag fused to the 3' end of the PfCINCH ORF (pRR209) was linearized by digestion, purified, and co-transfected with 75 μg Cas9 targeting plasmid into the P. falciparum 3D7 strain. One day post transfection, drug pressure was applied with 2.5 nM WR99210 (Jacobus Pharmaceuticals). Following verification of PfCINCH-mScarlet integration, PfCINCH-mScarlet parasites were transfected with 100 μg HDR plasmid containing the mNeonGreen fluorescent tag fused to the 3' end of the PfPPP8 ORF (pAM166), linearized by digestion, and 75 μg Cas9 targeting plasmid. One day post transfection, additional drug pressure was applied with 2.5 nM Blastocidin (Research Products International).

PfPPP8 depletion
In all assays comparing PfPPP8-sufficient and PfPPP8-deficient parasites, PfPPP8-smV5 Tet schizonts were purified by density centrifugation with 60% Percoll and washed twice in ATc-free complete RPMI. Schizonts were then replated into fresh erythrocytes and allowed to reinvade for 2 h. Following reinvasion, new rings were purified and synchronized by incubation with 5% (w/v) sorbitol synchronization and washed twice again in ATc-free complete RPMI. The resulting culture was then split 1:2, with half of the synchronized rings being added to RPMI with or without 500 nM ATc.

Replication assay
Synchronized PfPPP8-smV5 Tet (±ATC) was diluted to 0.2% parasitemia and 1% hematocrit and plated in triplicate. 100 uL of culture from each well was collected on days 1, 3, 5, and 7, washed with PBS, and resuspended in a 1:1000 dilution of SYBR green (Invitrogen) in 0.5% bovine serum albumin (BSA) in PBS. Parasites were incubated in the solution for 20 min at room temperature, then washed in 0.5% BSA in PBS and resuspended in filtered PBS. The proportion of infected cells was then determined by flow cytometry, utilizing the CellQuest Pro program for the collection and FlowJo X and GraphPad Prism 9 for analysis of results.

Immunofluorescence
Dried blood smears were fixed with 4% paraformaldehyde for 10 min, then rinsed 3× in PBS. Following fixation and washing, parasites were permeabilized with 0.1% Triton X-100, diluted in PBS, for 10 min, and washed again in 1× PBS 3× for 3 min. Blocking solution (3% (w/v) BSA in PBS) was added to slides for 1 h at room temperature or overnight at 4°C. Primary antibodies were diluted in blocking solution and added to slides for 1 h at room temperature or overnight at 4°C. Following primary antibody incubation, slides were incubated 3× for 5 min in 1× PBS. Secondary antibodies, diluted in 0.5% BSA-PBS, were then added to slides for 45 min at room temperature. Slides were washed again for 5 min, 3× in 1× PBS, then incubated with Hoechst 33342 diluted at 1:5000 in PBS. After rinsing three more times with 1× PBS, coverslips were mounted with VectaShield Vibrance. For the anti-CrCen3 immunofluorescence images, a modified method was used. Parasites were allowed to settle on poly-D-lysine coated coverslips, fixed with 4% paraformaldehyde, and then processed as described above. Cells were visualized on a Zeiss LSM980 with Airyscan2 for super-resolution microscopy, or on a Nikon N-SIM S microscope. In the Zeiss LSM980 a 63× objective with a numerical aperture of 1.4 was used; in the Nikon N-SIM S, a 100× objective with a numerical aperture of 1.35 was used. Dilutions for primary antibodies are: mouse anti-V5 1:500, rabbit anti-BCP1 1:250, rabbit anti-PfMORN1 1:1000, rabbit anti-PfGAP45 1:5000, mouse anti-PfMSP1 1:500, mouse anti-PfAMA1 1:200, mouse anti-CrCen 1:500, mouse anti-Myc 1:100, rabbit anti-Myc 1:200, rabbit anti-HaloTag 1:500.

Image processing
Display. For most of the Z-Stack images taken on the Zeiss LSM980 with AiryScan or Nikon N-SIM S microscopes and processed in ImageJ, each image was loaded into ImageJ and brightness and contrast were modulated solely by using the reset command (autoscale) on the brightness and contrast panel. This command restores the original brightness and contrast settings, setting the display range to the full pixel value of the image, and rendering processed images as similar as possible to native images. For certain images (listed in the relevant figures), the intensity values for each channel were matched to those of a previously "autoscaled" parasite using the SET command on FIJI.
Measurements. For measurements of circularity, each visible ring of the BC was traced using the FIJI freehand selection tool and the area and perimeter of each were measured. Circularity is defined as 4 × pi × area/perimeter 2 . Therefore, we then took the recorded area and perimeter and plugged them into a function written on RStudio to calculate circularity. A shape becomes more circular as the circularity approaches 1.
For measurements of ring diameter, each visible ring of the BC was traced using the FIJI freehand selection tool and an ellipse was fit to the freehand tracing using the set measurements and measure tools. The major diameter for each ellipse was reported. Fragmented rings that began to appear later in the PfPPP8-smV5 Tet -ATC condition were measured similarly if the break between two pieces was less than or equal to 0.4 microns and if there was no ambiguity as to what ring the fragment belonged to.
For measurements of average intensity values of fluorescent parasites, a rectangular region of interest was drawn precisely around the relevant parasite's maximum-projection image using the rectangular draw tool so each of the four sides "touched" the BC. This ROI was re-drawn at each time point so each of the four sides maintained contact with the BC; it shrank as the BC contracted and expanded as the BC expanded. The min&max gray value as well as the mean gray value were then measured using FIJI's set measurement and measure tools. The minimum gray value of each box, which stayed consistent throughout measurement, was subtracted from the mean gray value for each time point to subtract the background fluorescence. The mean gray value-min gray value for each fluorescent channel per parasite per time point was then divided by the original mean gray value-min gray value so the relative change in fluorescence could be graphed.

Transmission electron microscopy
Synchronized late PfPPP8-smV5 Tet schizonts, with and without ATC, were treated with 1 mM E64 at 48 hpi for 3 h. Parasites were then Percoll-purified and resuspended in fixative (2.5% paraformaldehyde, 5% glutaraldehyde, 0.06% picric acid in 0.2 M cacodylate buffer). Fixed parasites were prepared as standard for TEM and visualized on a JEOL 1200EX electron microscope.

Live cell microscopy
For PfPPP8 depletion progression movies (Fig. 3), PfPPP8-smV5 Tet -PfBLEB-HaloTag parasites were cultured in a variation of the high parasitemia method detailed in Radfar et al. 82 . To remove ATC, a high schizontemia culture was tightly synchronized as described for washouts. Upon progression to the schizont stage, in the following cycle, 200 uL of a mixed culture of schizonts from each condition was spun down and resuspended in 50 uL of media. The JaneliaFluor 647 Halo-Tag ligand was added at a concentration of 800 nM and allowed to settle on one quadrant of a conconavalin A-coated ibidi/cellview glass bottom dish. After 30 min, excess/nonbound red blood cells and the HaloTag ligand were washed off with three washes of PBS, then each quadrant was filled with phenol red free RPMI, with Trolox added to a concentration of 0.5 mM to minimize bleaching and oxidative damage. Several different fields were selected based on parasite density and stage of schizont over 6.5 h, using the 647 laser at 38% laser power and 400 ms exposure, with 2 × 2 binning (500 ms exposure and no binning for the supplementary repeat). Parasites were visualized on a Nikon TiEclipse equipped with confocal imaging. For measurement, resulting files were processed in FIJI, to produce supplementary movies, resulting files were processed in ARIVIS 4D.
For PfPPP8-PfCINCH timeline movies, PfCINCH-mNeonGreen-PfPPP8-mCherry parasites were cultured in a variation of the high parasitemia method detailed in Radfar et al. 82 . 200 uL of a mixed culture of schizonts from each condition was spun down, resuspended in 50 uL of media, and allowed to settle on one quadrant of a conconavalin A-coated ibidi/cellview glass bottom dish. After 30 min, excess/nonbound red blood cells were washed off with three washes of PBS, then each quadrant was filled with phenol red free RPMI, with Trolox added to a concentration of 0.5 mM to minimize bleaching and oxidative damage. Several different fields were selected based on parasite density and stage of schizont over 6.5 or 9 h, using the 488 laser at 13% laser power and 200 ms exposure, with 2 × 2 binning, and the 561 laser at 31% laser power, 400 ms exposure, and 2 × 2 binning. Parasites were visualized on a Nikon TiEclipse equipped with confocal imaging. For measurement, resulting files were processed in FIJI, to produce supplementary movies, resulting files were processed in ARIVIS 4D.

Expansion microscopy
For expansion microscopy, the protocol established by Absalon et al. was followed. Synchronized PfPPP8-smV5-PfCINCH-smMyc schizonts were purified via density centrifugation with 60% Percoll. Isolated schizonts were allowed to settle on poly-D-lysine coated coverslips for 20-30 min at 37°C. Parasites were fixed with 4% PFA for 20 min at 37°C, washed 3× with PBS, and fixed slips were incubated with FA/AA overnight at 37°C. Gel polymerization was performed the following morning; in ice, TEMED and APS were added to a previously made monomer solution and the coverslips were placed over a drop of the TEMED/APS/Monomer Solution mixture in a gelation chamber kept at -20°C for at least 15 min beforehand. After 5 min of incubation on ice, the chamber was incubated at 37°C for an hour. Post incubation, coverslips/gels were placed in 1 mL of denaturation buffer and incubated with denaturation buffer for 15 min with agitation. After gel detachment, gels were incubated for 90 more min in a 1.5 mL Eppendorf tube filled with denaturation buffer. After this incubation, denaturation buffer was removed and gels were placed in a 10 cm dish filled with ddH 2 O, which was switched out after 30 min. After overnight incubation in ddH 2 O, gels were washed in PBS two times for 15 min each then incubated in 3% BSA-PBS for 30 min at RT. Gels were then incubated in 1 mL of 3% BSA-PBS with primary antibodies (mouse anti-Myc, 1:100 or rabbit anti-Myc, 1:200; mouse anti-V5, 1:250) overnight at 4°C. The next morning, gels were washed three times with 2 mL 0.1% Tween20 in PBS for 10 min at room temperature with agitation. After washes, gels were incubated in 1 mL of PBS with secondary antibodies including NHS-Ester (647 and 405, 1:100) and Hoechst (1:5000), protected from light. After 2:30, gels were washed three more times with 2 mL 0.1% PBS + 0.1% Tween20 as previously, then placed in a 10 cm dish filled with ddH 2 O. Water was replaced after 30 min and gels were allowed to expand overnight before imaging on a Zeiss laser-scanning confocal 980 with Airyscan2.

Immunoprecipitation
PfPPP8-smV5 Tet -PfCINCH-smMyc and parental (Pf3D7) lines were both tightly synchronized by density centrifugation with percoll followed by treatment with 5% (w/v) sorbitol after reinvasion, then expanded to 250 mL. When parasites reached 3% schizontemia, they were stalled with 2.5 uM Protein Kinase G inhibitor C1 until roughly 50 hpi. RBCs were lysed with 0.05% saponin in PBS with protease inhibitors (Sig-maFast Protease Inhibitor Cocktail, Sigma). Parasite pellets were then lysed in ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) with protease inhibitors (SigmaFast Protease Inhibitor Cocktail) for 30 min on ice. Following incubation, samples were sonicated twice at 20% amplitude for 30 s and centrifugated to remove insoluble material. The supernatant from this centrifugation was added to magnetic anti-V5 and anti-Myc beads (Pierce Anti-c-Myc magnetic beads, Product #88842, MBL Anti-V5-tag magnetic beads, Product #M167-11) and incubated overnight at 4°C. Beads were then washed three times with RIPA + protease inhibitors, then twice with PBS plus protease inhibitors to remove detergents. They were then resuspended in 50 uL 5 mM ammonium bicarbonate and submitted to Harvard's Mass Spectrometry Core for on bead digestion and mass spectrometry analysis. Mass spectrometry results were analyzed by comparing the number of unique and total peptides and the sum intensities for each obtained protein between the experimental (PfPPP8-smV5 Tet -PfCINCH-smMyc) and control (3D7) lines. Resulting proteins were characterized as potential hits by using Microsoft Excel to filter out proteins present in the control samples and identify proteins present in both V5 and Myc samples.

Quantitative western blotting
Parasites were isolated by lysing RBCs in 0.05% saponin in PBS with protease inhibitors (SigmaFast Protease Inhibitor Cocktail). Pelleted parasites were washed with PBS until the supernatant was clear; samples were then boiled in Laemmli buffer for 5 min (for PfMyoJ-smV5 Tet parasites only: incubated at 60°C for 10 min). The equivalent of 1 mL of culture at 1% schizontemia was run on a 4-20% Tris-glycine-sodium dodecyl sulfate gel and transferred to either a polyvinylidene fluoride or nitrocellulose membrane. Membranes were blocked in Licor Odyssey blocking buffer, incubated with primary antibody, and then incubated in secondary antibodies diluted in blocking buffer. Following TBST-washing, Membranes were scanned on a Licor Odyssey CLx imager system and quantified using volumetric measurement of fluorescence intensity in LiCor Image Studio 4.0. Uncropped blot images are provided in the Source Data file. Dilutions of primary antibodies were: anti-V5 1:1000, anti-MORN1 1:2000, anti-H3 1:2500, anti-LDH 1:1000, anti-Myc 1:1000. Uncropped western blot images are provided in the Source Data file.

Statistics and reproducibility
For all single or few time point imaging experiments, three biological replicates were performed. Two biological replicates were performed for imaging via electron microscopy. For growth curve analysis, two biological replicates with three technical replicates per run were performed. For all immunoblotting experiments, three or more biological replicates were performed (four biological replicates to quantify the extent of PfPPP8 knockdown). For all live cell experiments, three biological replicates were performed. For all time course fixed-cell immunofluorescence or immunoprecipitation experiments requiring tight synchronization of parasites, close monitoring, and/or large volumes of synchronous parasite culture, two biological replicates were performed. For all recombinant protein experiments, two biological replicates with three technical replicates per biological replicate were performed.